Background: Friedreich ataxia (FRDA), the most common form of recessive ataxia, is due to reduced levels of frataxin, a highly conserved mitochondrial iron-chaperone involved in iron-sulfur cluster (ISC) biogenesis. Most patients are homozygous for a (GAA)(n) expansion within the first intron of the frataxin gene. A few patients, either with typical or atypical clinical presentation, are compound heterozygous for the GAA expansion and a micromutation.

Methodology: We have developed a new strategy to generate murine cellular models for FRDA: cell lines carrying a frataxin conditional allele were used in combination with an EGFP-Cre recombinase to create murine cellular models depleted for endogenous frataxin and expressing missense-mutated human frataxin. We showed that complete absence of murine frataxin in fibroblasts inhibits cell division and leads to cell death. This lethal phenotype was rescued through transgenic expression of human wild type as well as mutant (hFXN(G130V) and hFXN(I154F)) frataxin. Interestingly, cells expressing the mutated frataxin presented a FRDA-like biochemical phenotype. Though both mutations affected mitochondrial ISC enzymes activities and mitochondria ultrastructure, the hFXN(I154F) mutant presented a more severe phenotype with affected cytosolic and nuclear ISC enzyme activities, mitochondrial iron accumulation and an increased sensitivity to oxidative stress. The differential phenotype correlates with disease severity observed in FRDA patients.

Conclusions: These new cellular models, which are the first to spontaneously reproduce all the biochemical phenotypes associated with FRDA, are important tools to gain new insights into the in vivo consequences of pathological missense mutations as well as for large-scale pharmacological screening aimed at compensating frataxin deficiency.

pone-0006379-g006: ROS determination and antioxidative enzymes activities in wild-type hFXN, mutant hFXNG130V and hFXNI154F clones.A. FACS analysis of cells incubated with DHR123. The dashed curve represents the autofluorescence of fibroblasts cells without DHR123 treatment. The black curve represents the observed fluorescence in cells in endogenous conditions and the grey curve is the fluorescence induced after hydrogen peroxide incubation (10 µM; 30 min). Experiments were done in triplicate on 3 clones of hFXN, hFXNG130V and hFXNI154F. Results show one representative diagram from one clone for hFXN and hFXNG130V, and 2 clones for hFXNI154F. B. Enzymatic actvities of catalase (grey bars) and glutathione reductase (black bars) were determined spectrophotometrically. Activities are presented as the percentage of WT hFXN clones activity. Data are represented as mean + SD. * p<0.05. C. Catalase protein was detected using an anti-catalase antibody and compared to GAPDH as a loading control.

Mentions:
A recognized characteristic of frataxin deficiency is the increased susceptibility to induction of endogenous or exogenous oxidative stress in FRDA patient cells, and in yeast, C.elegans and Drosophila models [4], [34]. To investigate the oxidative status of the newly developed cell models, we monitored the presence of intracellular ROS (peroxides and peroxynitrite anion) using the dihydrorhodamine 123 probe. Comparison of oxidation-induced fluorescence of the probe by FACS between hFXN, hFXNG130V and hFXNI154F clones revealed no difference in the production of ROS in normal culture conditions (Fig. 6A). However, the hFXNI154F clones were more sensitive to exogenous stress, as a treatment with 10 µM hydrogen peroxide caused a drastic change in fluorescence level in hFXNI154F clones compared to moderate effects on hFXN and hFXNG130V clones (Fig. 6A). Furthermore, the hFXNI154F-2C1 clone had a lower susceptibility to stress than the hFXNI154F-1D3 clone since the fluorescence peak in the later was fully shifted. This hFXNI154F-2C1 clone appeared globally less affected than the two others hFXNI154F clones (1D3 and C6). Interestingly, both hFXNG130V and hFXNI154F clones displayed a significantly reduced catalase activity (44% and 51%, respectively, Fig. 6B) that correlated with a decrease in catalase expression at the protein level (Fig. 6C) and at the mRNA level (not shown). On the other hand, no significant change was observed in the activity of glutathione reductase (Fig. 6B). Furthermore, no difference in the mitochondrial superoxide dismutase (Sod2) expression at the protein level (Fig. 2A) or at the RNA level (data not shown) was observed. Overall, these results suggest that the combination of iron deregulation with a deficit in the antioxidant enzyme catalase could explain the susceptibility to exogenous oxidative stress observed for the hFXNI154F clones.

pone-0006379-g006: ROS determination and antioxidative enzymes activities in wild-type hFXN, mutant hFXNG130V and hFXNI154F clones.A. FACS analysis of cells incubated with DHR123. The dashed curve represents the autofluorescence of fibroblasts cells without DHR123 treatment. The black curve represents the observed fluorescence in cells in endogenous conditions and the grey curve is the fluorescence induced after hydrogen peroxide incubation (10 µM; 30 min). Experiments were done in triplicate on 3 clones of hFXN, hFXNG130V and hFXNI154F. Results show one representative diagram from one clone for hFXN and hFXNG130V, and 2 clones for hFXNI154F. B. Enzymatic actvities of catalase (grey bars) and glutathione reductase (black bars) were determined spectrophotometrically. Activities are presented as the percentage of WT hFXN clones activity. Data are represented as mean + SD. * p<0.05. C. Catalase protein was detected using an anti-catalase antibody and compared to GAPDH as a loading control.

Mentions:
A recognized characteristic of frataxin deficiency is the increased susceptibility to induction of endogenous or exogenous oxidative stress in FRDA patient cells, and in yeast, C.elegans and Drosophila models [4], [34]. To investigate the oxidative status of the newly developed cell models, we monitored the presence of intracellular ROS (peroxides and peroxynitrite anion) using the dihydrorhodamine 123 probe. Comparison of oxidation-induced fluorescence of the probe by FACS between hFXN, hFXNG130V and hFXNI154F clones revealed no difference in the production of ROS in normal culture conditions (Fig. 6A). However, the hFXNI154F clones were more sensitive to exogenous stress, as a treatment with 10 µM hydrogen peroxide caused a drastic change in fluorescence level in hFXNI154F clones compared to moderate effects on hFXN and hFXNG130V clones (Fig. 6A). Furthermore, the hFXNI154F-2C1 clone had a lower susceptibility to stress than the hFXNI154F-1D3 clone since the fluorescence peak in the later was fully shifted. This hFXNI154F-2C1 clone appeared globally less affected than the two others hFXNI154F clones (1D3 and C6). Interestingly, both hFXNG130V and hFXNI154F clones displayed a significantly reduced catalase activity (44% and 51%, respectively, Fig. 6B) that correlated with a decrease in catalase expression at the protein level (Fig. 6C) and at the mRNA level (not shown). On the other hand, no significant change was observed in the activity of glutathione reductase (Fig. 6B). Furthermore, no difference in the mitochondrial superoxide dismutase (Sod2) expression at the protein level (Fig. 2A) or at the RNA level (data not shown) was observed. Overall, these results suggest that the combination of iron deregulation with a deficit in the antioxidant enzyme catalase could explain the susceptibility to exogenous oxidative stress observed for the hFXNI154F clones.

Background: Friedreich ataxia (FRDA), the most common form of recessive ataxia, is due to reduced levels of frataxin, a highly conserved mitochondrial iron-chaperone involved in iron-sulfur cluster (ISC) biogenesis. Most patients are homozygous for a (GAA)(n) expansion within the first intron of the frataxin gene. A few patients, either with typical or atypical clinical presentation, are compound heterozygous for the GAA expansion and a micromutation.

Methodology: We have developed a new strategy to generate murine cellular models for FRDA: cell lines carrying a frataxin conditional allele were used in combination with an EGFP-Cre recombinase to create murine cellular models depleted for endogenous frataxin and expressing missense-mutated human frataxin. We showed that complete absence of murine frataxin in fibroblasts inhibits cell division and leads to cell death. This lethal phenotype was rescued through transgenic expression of human wild type as well as mutant (hFXN(G130V) and hFXN(I154F)) frataxin. Interestingly, cells expressing the mutated frataxin presented a FRDA-like biochemical phenotype. Though both mutations affected mitochondrial ISC enzymes activities and mitochondria ultrastructure, the hFXN(I154F) mutant presented a more severe phenotype with affected cytosolic and nuclear ISC enzyme activities, mitochondrial iron accumulation and an increased sensitivity to oxidative stress. The differential phenotype correlates with disease severity observed in FRDA patients.

Conclusions: These new cellular models, which are the first to spontaneously reproduce all the biochemical phenotypes associated with FRDA, are important tools to gain new insights into the in vivo consequences of pathological missense mutations as well as for large-scale pharmacological screening aimed at compensating frataxin deficiency.